Summary We recently established that microsatellite instable (MSI) colorectal cancer (CRC), which have a DNA mismatch repair deficiency and consequently accumulate high density of somatic mutations, exhibits a robust IFN?hiPD-1hi CD8+ T cell infiltration associated with myeloid expression of the PD-1 ligand, PD- L1. Further identification of additional T cell checkpoints, including CTLA-4 and LAG-3, suggest that MSI CRC triggers an endogenous anti-tumor immune response that is counter-regulated by the coordinate expression of a variety of immunoregulatory ligands. In support of this hypothesis, a clinical trial initiated at Johns Hopkins Hospital (JHH) found that MSI metastatic CRC (mCRC) patients demonstrated a 62% objective response rate (ORR) to anti-PD-1 immunotherapy. Importantly, whereas we identified a subset (14%) of primary microsatellite stable (MSS) CRC which exhibits also a robust Th1/Tc1-type inflammation, a similar fraction of MSS mCRC was sensitive to anti-PD-1 therapy (11% progression-free disease) suggesting that the efficiency of anti- PD-1 may be associated with the presence of an endogenous immune response in MSI but also a fraction of immunogenic MSS CRC. These discoveries raise a number of questions that bear on advances in immunotherapy: What are the immunologic determinants in the tumor microenvironment that distinguish a responder from non responder CRC patients to anti-PD-1? Do the somatic mutations of MSS CRC generate neoantigens (mutation-associated neoantigens or MANAs) that are recognized by the infiltrating T cells? Does the density over the nature of the mutations drive the intensity of the endogenous immune response? And finally since MSI CRC represents only 5% of the metastatic CRC, can we identify a larger population of CRC patient exhibiting an endogenous anti-tumor immune response that will benefit from checkpoint blockade therapeutic approach? To answer these questions, we propose three Aims in this proposal: 1) Identifying outlier MSS CRC that exhibit a MSI-like immune signature; 2) Testing that MSS and MSI CRC-infiltrating CD8+ T cells recognize neoantigens; and 3) Validate that MANA-specific T cells are unleashed in mCRC responding to anti-PD-1. In finally, this proposal will define the correlates of the immune response to neoantigens in CRC and will test the hypothesis that such biomarkers delineate MSS metastatic CRC patients who could benefit from anti- PD1 therapy.